Expression and biochemical characterization of GumI, a bacterial ß-mannosyltransferase

SALINAS, SR; ABDIÁN, PL; KAMPEL, MA; BARRERAS, M; IELPI

Mendoza, Argentina

Congreso; XLIV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2008

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0pt; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:ES-AR; mso-fareast-language:EN-US;} @page Section1 {size:595.45pt 841.7pt; margin:72.0pt 90.0pt 72.0pt 90.0pt; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Xanthomonas campestris is a Gram negative bacterium that produces an exopolysaccharide known as xanthan gum. Xanthan is involved in a variety of biological functions, including pathogenesis, and is widely applied in food and non food industry. Although the genetics and biosynthetic process of xanthan are well documented, the enzymatic components remain very poorly characterized. We describe here the expression, purification and functional characterization of the gumI gene product, an essential protein for xanthan synthesis. Complementation studies and immunoblots showed correspondence between the proposed gene and the 39-kDa protein product, and that it is membrane-associated. The membrane association was independent of the remaining Gum proteins. The protein was overexpressed in Escherichia coli, solubilized and purified in active and stable form using Ni-chelating and size-exclusion columns. The purified protein catalyzed the transfer of a mannosyl residue from GDP-mannose to glucuronic acid-β-1,2-mannose-α-1,3-glucose-β-1,4-glucose-P-P-polyisoprenyl with formation of a mannose-β-1,4-glucuronic acid linkage. The enzyme shows no homology with any of the 91 currently known carbohydrate-active enzyme families. The procedure described here produced good yield of pure protein through two chromatographic steps, suitable for further structure–function studies.